Study Of IL-9 And CD39 In Malignant Tumor Growth

Tumor is a class of the most common diseases and malignant tumor is the major disease which seriously hazard human health. With the development of study on operation, chemotherapy and radiotherapy, the long-term survival of malignant tumor patients has been improved. However, some patients still suffer from tumor recurrence or metastases.Non-Hodgkin lymphoma (NHL) is a diverse group of lymphoid malignancies, many of which remain incurable. B-cell NHL account for over 90% of lymphoid neoplasms worldwide with about 4% of new cases being diagnosed each year. The most common types are follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), which make up 50% of the NHL. Melanoma is a malignant tumor of melanocytes which originally from skin, as also as eyes and nasal cavity. Melanoma is less common than other skin cancers. However, it is much more dangerous and causes the majority (75%) of deaths related to skin cancer. Increasing evidence suggests that the tumor microenvironment plays an important role in determining not only the severity of disease, but also responsiveness to therapy.T regulatory cells (Treg) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. CD4+CD25+Foxp3+ Treg cells are the most important subpopulation of Treg cells, which can secret series of cytokines, such as IL-10, TGF-P and CTLA-4. They play important roles in autoimmune diseases, transplantation immunity and tumor immunity. Mast cells (MCs) are a critical immune cell type, which can also function as immune regulatory cells. The association between MCs and human cancer has emerged and many studies have highlighted a correlation between the amount of tumor-infiltrating MCs and the degree of tumor aggressiveness (ie, grade) and dissemination (ie, stage), suggesting a key role of MCs in tumor growth. IL-9 has been implicated as a crucial factor in the regulation of MC recruitment and their effector functions. Some studies support the concept that IL-9 represents the functional link through which activated Treg cells recruit and activate MCs to mediate regional immune suppression in a skin transplantation model. Recently, it is reported that Treg/MC interaction is of critical importance for limiting endogenous inflammatory disease and the anti-inflammatory effects of Tregs critically depend on IL-9-mediated attraction of MCs into kidney-draining lymph nodes in a model of nephrotoxic serum nephritis. However, the roles of complicated interactions among Treg cells, MCs and IL-9 in tumor progression are still unclear.Adenosine triphosphate (ATP) mediates multiple physiological reactions and plays a crucial role in cellular metabolism, inclusive of roles in bioenergetics. Extracellular ATP acts on type 2 purinergic (P2) receptors to exert signaling effects. Intracellular ATP concentrations are typically of the order of 3 to 10 mM. Basal concentrations of extracellular ATP, in contrast, are considered to be around 10 nM. The latter levels are maintained by ectonucleotidases, which hydrolyze released ATP sequentially to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and further to adenosine. These ectoenzymes result in a 106-fold gradient for potential ATP efflux. Therefore, the release of a small amount of intracellular ATP could elicit a dramatic elevation of extracellular ATP concentration thereby impacting purinergic signaling.Anticancer chemotherapies directly induce tumor cell death. Dying tumor cells release mediators that signal cellular damage, including ATP. ATP has been recently identified as a novel danger signal emitted by dying tumor cells and is also released by immune cells. ATP is considered important for the efficient immune responses required for the successful anticancer therapies. ATP can also be released from the cytosol of necrotic cells, which are always present in the center of fast-growing tumors, such as in transplanted melanomas.CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the dominant ectonucleotidase expressed by endothelial cells (ECs) and Treg cells. When ATP appears in the extracellular space of tumor microenvironment, it is quickly metabolized by CD39 to AMP. Therefore, in Cd39 null mice, failure of removal of ATP released by necrotic tumor cells in the center of fast-growing tumors might cause acute increases in levels of local extracellular ATP, and result in killing of adjacent tumor cells. Thus, targeting the expression and/or ectoenzymatic activity of CD39 in combination with other chemotherapy regimens might provide a novel approach to cancer therapy.In this study, we will further elucidate the significance of IL-9 and CD39 in tumor progression by using molecular biology, cellular biology and series of immunology techniques. We will also explore the mechanism of tumor immune tolerance and search new immunotherapy methods to improve prognosis of malignant tumor patients.Part I Study of IL-9 in regulatory T cells and mast cells mediated immunosuppression in B-cell non-Hodgkin's lymphomaObjective:Non-Hodgkin's lymphoma (NHL) is a diverse group of malignancies, which originally from B and T lymphocytes. Immune system functional disorder is closely related to the genesis of lymphoma. The infiltration of regulatory T cells (Treg) and mast cells (MCs) in tumor tissue plays important roles in tumor immune tolerance. IL-9 is a crucial factor in the regulation of MC recruitment and their effector functions. In order to study the roles of IL-9 in Treg cells and MCs mediated immunosuppression in B-cell NHL, blood samples and tumor tissues were collected from 32 patients who were diagnosed with B-cell NHL for the first time at the Hematology Department in our hospital. Treg cells and MCs-related protein expressions in tumor tissues were examined by Western-blot and immunohistochemistry. IL-9 level in serum was measured by ELISA. Treg and MC numbers/HP were counted and compared with the main clinical features of the patients at diagnosis. The relationships among numbers of Treg cells, numbers of MCs, serum LDH andβ2-MG concentration of the patients were also analyzed. A murine model of lymphoma was established to explore the effects of IL-9 to Treg cells and MCs. Tumor sizes, expressions of Treg cells and MCs related genes were detected after IL-9 neutralization. To further explore the effects of IL-9 to Treg cells function, apoptosis and MC induction and function, Treg cells were isolated and their function and apoptosis were analyzed. And also, IL-9 was added into the culturing bone marrow cells. Bone-marrow-derived mast cells (BMMC) purity and functional genes expressions were detected.Material and Methods:1. Specimen collection2. Cell culture and animal experiments3. Protein extraction and Western Blot4. Immunohistochemistry (IHC)5. Serum collection and ELISA6. RNA extraction and RT-PCR7. BMMC culture and purity assay8. Treg cells and Teff cells isolation9. Treg cells culture and Teff cells proliferation assay10. FACS11. Statistical analysisResults:1. Expressions of Foxp3, CD117, and IL-9 proteins in B-cell NHL patientsFoxp3 and CD117 were up-regulated significantly in B-cell NHL involved tissues when compared with controls (Foxp3 optical density ratios:0.3084±0.0651 v. 0.1334±0.0546, P<0.001; CD117 optical density ratios:0.0551±0.0064 v. 0.0192±0.0072, P<0.001). Foxp3+ Treg cells and CD117+ MCs were observed in all cases. The topographic distribution of Treg cells within the tumor varied from case to case. CD117+ MCs aggregated within superficial parenchyma, especially around small blood vessels and lymph vessels. The presences of more Foxp3+ Treg cells and CD117+ MCs were found in tissues from B-cell NHL subjects compared with controls. Serum samples from patients with B-cell NHL contained detectable levels of IL-9 in a substantially higher frequency than the sera from healthy controls.2. Correlation between clinicohistologic features and Foxp3+ Treg and CD117+ MC numbers in biopsy samplesNo significant correlation was observed between Treg numbers and the main initial characteristics. When correlations of MC numbers and the main clinical features of the patients were analyzed, no significant correlation was observed between MC numbers and the main initial characteristics. However, more CD117+ MCs can be found in patients with detectable serum IL-9 than those in patients with undetectable serum IL-9.3. Correlation between Treg cells, MCs and markers of B-cell NHLThe number of Foxp3+ Treg cells was highly correlated with the number of MCs (rs=0.7083, P<0.01). In addition, positive correlations between numbers of Treg cells and serum LDH orβ2-MG concentration could be seen. Numbers of MCs also had significant positive relationships with serum LDH orβ2-MG concentration. 4. IL-9 favors tumor growth in murine lymphoma modelWhen compared with sera from normal BALB/c mice, higher IL-9 concentrations were found in sera from A20 inoculated mice (744.18±76.88 v. 388.94±54.74 pg/ml, P<0.01). In comparison with isotype control antibody treated mice, the neutralization of IL-9 with anti-mIL-9 blocking antibody induced a much slower tumor growth.5. IL-9 regulates expression of Treg cell and MC-related genes in tumor draining lymph nodes and then suppressive tumor microenvironmentWe examined Treg cell and MC-related gene expressions in tumor draining lymph nodes, including Foxp3, stem cell receptor (CD117), high-affinity IgE receptor (Fcer1a), mast cell protease 1(Mcpt1) and mast cell protease 5 (Mcpt5). All of them were up-regulated in isotype control antibody treated group compared with normal lymph nodes, while significantly down-regulated in anti-mIL-9 blocking antibody treated group.6. IL-9 can be produced by activated Treg cells, and IL-9 can modulate Treg cell function and apoptosisTreg cells, when activated by anti-CD3 and anti-CD28 antibody in vitro, could produce high levels of IL-9 and its concentration increased in a time-dependent manner. Treg cells and CD4+CD25- Teff cells were isolated and co-cultured. Teff cells proliferation was measured. Addition of rIL-9 significantly inhibited the proliferation of Teff cells through increasing the suppressive capacity of nTreg cells. In contrast, blockade of IL-9 signaling with an anti-mIL-9 neutralizing antibody reversed nTreg-mediated suppression, allowing Teff cells to proliferate.nTreg cells were cultured in a serum-free medium with or without rIL-9. We found that nTreg cells died quickly while rIL-9 addition partially rescued Treg cells from apoptotic cell death and significantly decreased the number of necrosis cells. Anti-mIL-9 neutralizing antibody could reverse the effects of rIL-9 to nTreg cells.7. Effects of IL-9 on IL-3 and SCF-dependent BMMC development and MC-related gene expressionBone marrow cells were cultured in medium containing rIL-3 and rSCF. The addition of rIL-9 to the culture gave rise to increase in the purity of BMMCs in the first three weeks. This observation was associated with increased expressions of MC-related genes, including CD117, Fcer1a, Mcpt1 and Mcpt5.Conclusion:Our study showed that Treg cells and MCs played important roles in immune-system which are associated with risks of B-cell NHL progress. IL-9 promotes tumor growth not only by affecting natural Treg cells functions and apoptosis, but also by promoting MCs functions and inductions in vivo. IL-9 contributes to Treg cells and MCs related immunosuppression in B-cell NHL. Pharmacological or targeted inhibition of IL-9 activity may find utility as an adjunctive in B-cell NHL therapy. Part II Study of vascular CD39/ENTPD1 in tumor growthObjective:Adenosine triphosphate (ATP) mediates multiple physiological reactions and plays a crucial role in cellular metabolism, inclusive of roles in bioenergetics. Extracellular ATP acts on type 2 purinergic (P2) receptors to exert signaling effects. The release of a small amount of intracellular ATP could elicit a dramatic elevation of extracellular ATP concentration thereby affecting purinergic signaling. Anticancer chemotherapies directly induce tumor cell death. Dying tumor cells release mediators that signal cellular damage, including ATP. CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the dominant ectonucleotidase expressed by endothelial cells (ECs) and Treg cells. When ATP appears in the extracellular space of tumor microenvironment, it is quickly metabolized by CD39 to AMP. In our study, we analyzed the effects of ATP to tumor growth and related mechanisms to further explore the roles of vascular CD39 in ATP-mediated tumor inhibition.Material and Methods:1. Animal research and cell culture2. Luc-B16/F10proliferation and cell viability assay3. Liver sinusoidal endothelial cells (LSECs) isolation, culture and purity assay4. ATP treated luc-B16/F10 cells were cultured with P2R antiagonist or LSEC5. Protein extraction and Western Blot6. RNA extraction, RT-PCR and real-time RT-PCR7. FACS8. Tumor supernatant preparation9. Measurement of ATP levels in biological samples10. Immunocytohistochemistry and immunofluorescence11. Thin Layer Chromatography (TLC) Analysis12. Statistical analysisResults:1. Antiproliferative functions of ATP are mediated via P2X7 receptor Luciferase-expressing B16/F10 (luc-B16/F10) cells were used for the present study. Cell proliferation was inhibited by exposure (16 hours) of ATP in a concentration-dependent manner. BzATP (synthetic non-hydrolyzable and potent ATP analogue) had more potent inhibitory effects on melanoma cell proliferation, as expected. Similar inhibitory effects of extracellular ATP on other MCA38 colon cancer cells were also observed.Luc-B16/F10 cells were incubated with P2 antagonists including KN-62 (to P2X7), MRS-2500 (to P2Y,), and suramin (non-selective to P2Rs), together with ATP (2.5 mM) for 16 hours. Changes in cell proliferation were then evaluated. Coincubation of cells with KN-62 decreased the extent of ATP-induced inhibition on melanoma cell proliferation in a dose-dependent manner with noted cytotoxicity of 2.5μM KN-62 alone. MRS-2500, or suramin, failed to block the inhibitory effects of ATP.Our Western blot analysis showed P2X7 was expressed in luc-B16/F10 cells. Taken together, our data suggest that P2X7 receptor mediates, at least in part, the antiproliferation action of ATP in B16 melanoma cells.2. ATP promotes tumor cell death through P2X7 receptorWe also found ATP could induce cytotoxicity on melanoma cells. In situ cellular analysis demonstrated that melanoma cell death with decreases in cell confluency and cell count was triggered on ATP stimulation, in a dose-dependent manner. Similar effects were observed in MCA38 colon cancer cells albeit with different sensitivity to ATP.Luc-B16/F10 cells treated with ATP alone or together with KN-62, or MRS-2500, or suramin were stained simultaneously with both FITC-annexin V and PI. Induction of both apoptosis and necrosis were observed in cells exposed to ATP in a time-dependent manner. In addition, ATP-stimulated apoptotic/necrotic cell death could be blocked by coincubation with a P2X7 antagonist, KN-62, but not by coincubation of MRS-2500 or suramin.The levels of cleaved caspase-3 and caspase-9 increased over time after exposure to ATP. These observations were further confirmed by Western blot analysis using anti-cleaved caspase-3 and caspase-9 antibodies. Furthermore, coincubation of cells with KN-62 blocked ATP-induced cleavage of caspase-3 and caspase-9.Quantitative real-time PCR revealed that treatment with ATP for 16 hours significantly increased the mRNA expression level of P2X7, whereas cotreatment with KN-62 abrogated the increase in P2X7 expression. These findings indicate that ATP-induced cell death in B16 melanoma cells is associated with both apoptosis and necrosis and is at least partly mediated through the P2X7 receptor.3. Apyrase (soluble NTPDase) or vascular cell CD39 expressed by LSECs abrogates/reverses anti-tumor activity of ATPTumor cell growth inhibition triggered by ATP was completely abrogated by coincubation of cells with apyrase. The rescue of tumor cells by apyrase was dose dependent. CD39 and CD73 are the major ectonucleotidases expressed by LSECs. WT LSECs attenuated the inhibitory effects of ATP on tumor cell growth, whereas Cd39 null LSECs did not retain this capacity. Interestingly, growth inhibition by ATP was reversed by Cd73 null LSECs to a greater extent, when compared with WT LSECs. The rescue observed with WT and Cd73 null LSECs was dose dependent. We also noted that extracellular ATP inhibited growth of LSECs.TLC analysis using ADP-C14 as substrate showed ADP was first hydrolyzed to AMP, and then to adenosine by WT LSECs. Adenosine was further degraded to hypoxanthine by WT LSECs. Cd73 null LSECs could only generate AMP but not adenosine. Cd39 null LSECs and luc-B16/F10 cells had minimal nonspecific ectonucleotidase activity, in contrast to WT LSECs.4. Defective angiogenesis is associated with heightened tumor necrosis and increased P2X7 expression in Cd39 null tumor-bearing miceInjured tumor cells could release endogenous mediators that directly result in cellular damage of contiguous/adjacent tumor cells. We found that melanoma cell proliferation was inhibited by the addition of tumor supernatants in a concentration-dependent manner. Dramatic increases in ATP levels were also noted in these tumor supernatants compared with the prewash media. However, coincubation with apyrase alone failed to rescue the growth inhibitory effects triggered by these crude tumor supernatants as previously noted with exogenous ATP. These data suggest that other cytotoxic constituents besides nucleotides contribute to the tumor killing activity of supernatants.We stained liver tumor sections using anti-CD31and anti-CD39 antibodies. We observed that CD39 was expressed on tumor-associated endothelial cells (ECs) in WT livers. In contrast, in Cd39 null tumor-bearing livers, lack of CD39 expression was associated with defective angiogenesis and larger areas of necrosis within the centers of tumors. Immunofluorescent staining on tumor sections further showed that protein expression of P2X7 was increased on melanoma cells in Cd39 null tumor-bearing livers compared with WT livers.Conclusion:High levels of extracellular ATP inhibit tumor cell proliferation in a dose-dependent manner through P2X7 receptor. And also, it can promote tumor cell death in a dose-dependent manner through P2X7 receptor. Apyrase or vascular cell CD39 expressed by LSECs abrogates/reverses antitumor activity of ATP by scavenging extracellular ATP. Defective angiogenesis is associated with heightened tumor necrosis and increased P2X7 expression in Cd39 null tumor-bearing mice. Besides ATP, injured tumor cells could release endogenous mediators that result in cellular damage of contiguous/adjacent tumor cells. The antitumor activities of ATP are dose-dependent and can be amplified by inhibition of ectonucleotidase, such as CD39, open new avenues for investigation in cancer management.